Prevalence and mechanisms of aminoglycoside resistance among drug-resistant Pseudomonas aeruginosa clinical isolates in Iran

Background Aminoglycosides have been a cornerstone of the treatment of nosocomial infections caused by Pseudomonas aeruginosa for over 80 years. However, escalating emergence of resistance poses a significant challenge. Therefore, this study aimed to investigate the prevailing patterns of aminoglycoside resistance among clinical isolates of P. aeruginosa in Iran; as well as the underlying resistance mechanisms observed in patients referred to Ardabil hospitals. Methods A total of 200 isolates from five hospitals were evaluated. The resistance profiles of P. aeruginosa isolates to tobramycin, amikacin, and netilmicin were determined using the disk diffusion method. The capacity of aminoglycoside-resistant isolates to form biofilms was assessed through a phenotypic assay, and the results were confirmed using the gene amplification technique. The presence of genes associated with aminoglycoside resistance was detected using polymerase chain reaction (PCR). Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression levels of genes encoding the MexXY-OprM efflux pump and PhoPQ two-component system (TCS). Results The prevalence of aminoglycoside-resistant P. aeruginosa isolates was 48%, with 94.7% demonstrating multidrug resistance (MDR). All aminoglycoside-resistant P. aeruginosa strains exhibited biofilm-forming capabilities and harbored all the genes associated with biofilm production. Among the nine genes encoding 16S rRNA methylase and aminoglycoside-modifying enzymes, three genes were detected in these isolates: aac(6’)-Ib (85.4%), ant(2’’)-Ia (18.7%), and aph(3’)-VI (3.1%). Additionally, all aminoglycoside-resistant P. aeruginosa isolates carried mexY and phoP genes, although the expression levels of mexY and phoP were 75% and 87.5%, respectively. Conclusion Given the considerably high prevalence of aminoglycoside-resistant P. aeruginosa strains, urgent measures are warranted to transition towards the use of novel aminoglycosides and to uphold vigilant surveillance of resistance patterns. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-024-09585-6.


Background
Hospitalized patients face a heightened risk of acquiring diverse hospital-acquired infections, among which Pseudomonas aeruginosa stands out as a prominent causative agent, particularly in intensive care units (ICUs) [1][2][3][4].Commonly prescribed antibiotics for combating P. aeruginosa infections in hospitals settings encompass β-lactams, fluoroquinolones, and aminoglycosides [5,6].However, effective eradication of this nosocomial pathogen from hospital environments and curbing mortality rates in severe infections present considerable challenges because of its propensity to develop resistance against a broad spectrum of antiseptics, disinfectants, and antibiotics.This has led to increased costs and prolonged hospital stay [3,7,8].
Aminoglycosides, known for their bactericidal properties and synergistic effects with β-lactams, are commonly employed in the treatment of various P. aeruginosa infections, notably pulmonary infections in patients with cystic fibrosis (CF) [8].However, the emergence of aminoglycoside-resistant P. aeruginosa strains has become a global concern since its initial recognition in the 1960s [8].The resistance of P. aeruginosa to aminoglycosides can be attributed to several mechanisms, including (1) alterations in outer membrane permeability facilitated by lipopolysaccharide (LPS) modifications mediated by the PhoPQ two-component system (TCS); (2) efflux systems such as MexXY-OprM; (3) ribosomal changes caused by 16S rRNA ribosomal methyltransferases; (4) production of antibiotic-inactivating enzymes such as aminoglycoside phosphotransferases (APH), aminoglycoside acetyltransferases (AAC), and aminoglycoside nucleotidyltransferases (ANT); and (5) biofilm formation [8,9].

Bacterial isolates
In this study, glycerol stocks of P. aeruginosa clinical isolates (n = 200) were utilized, sourced from five affiliated hospitals of Ardabil Medical University between June 2019 and May 2023.These isolates were previously identified through standard phenotypic and genotypic testing protocols.P. aeruginosa clinical isolates had been collected from inpatients and outpatients and duplicate isolates were excluded from the study.

Antimicrobial susceptibility testing
The resistance profiles of P. aeruginosa to three aminoglycosides (i.e., tobramycin, amikacin, and netilmicin) were assessed using the disk diffusion method, following the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI, 2024) [11].The disk diffusion procedure was conducted according to established protocols, with P. aeruginosa (ATCC 27,853) utilized as a control strain for consistency [4].Colistin resistance was previously done using the colistin agar test [10].

Biofilm formation assay
Biofilm formation ability was determined for aminoglycoside-resistance P. aeruginosa clinical isolates using the colorimetric microtiter plate, following the method previously described by Jabalameli et al. [12].In this procedure, the optical density (OD) of each distinct bacterial strain was measured at 590 nm using an ELISA reader and the results were recorded as ODs (the OD of a strain) ≤ ODc (the OD of the negative control) (no biofilm producer), ODc < ODs < 2× ODc (weak biofilm producer), 2× ODc < ODs < 4× ODc (moderate biofilm producer), and 4× ODc < ODs (strong biofilm producer).

Amplification of aminoglycosides resistance genes
The genes responsible for biofilm formation (i.e., algD, pslD, pelF, Ppgl, and PAPI-1 genes), 16S rRNA methylase (i.e., rmtA, rmtB, rmtC, rmtD, and armA genes), aminoglycoside-modifying enzymes (i.e., aac(6')-Ib, aac(6')-IIa, aph(3')-VI, and ant(2'')-Ia genes), efflux pump (mexY gene) and TCS (phoP gene) were detected using the polymerase chain reaction (PCR) method.Deoxyribonucleic acid (DNA) extraction from aminoglycoside-resistance P. aeruginosa strains was carried out using the boiling method.Each PCR reaction had a final volume of 25 µL and followed a standardized thermal cycling protocol: 1 cycle of initial denaturation at 95 °C for 5 min, followed by 30 to 34 cycles consisting of denaturation at 94 °C for 1 min, annealing at various temperatures (as specified in Table 1) for 1 min, and extension at 72 °C for 1 min.The oligonucleotide sequences of the primers used and the corresponding annealing temperatures for each gene are provided in Table 1.Sequencing of the PCR product of each amplified gene was conducted, and the sequences were utilized as positive control.
The accession numbers for these sequences are available in the NCBI GenBank repository (OR855380, OR855383 to OR855385, PP468580 to PP468582, ON920997, and OR855381).

Expression of the MexXY-OprM efflux pump and PhoPQ TCS
The quantitative reverse transcription PCR (qRT-PCR) technique was employed to quantify the expression levels of the genes encoding MexXY-OprM efflux pump (mexY gene) and PhoPQ TCS (phoP gene).Sixteen multidrug-resistant (MDR) P. aeruginosa clinical isolates were selected for this analysis.These MDR isolates also were extensively drug-resistant (XDR) or pandrug-resistant (PDR) or colistin-resistant bacteria.Total RNA extraction and cDNA synthesis were conducted using TRIzol ™ reagent (Bio Basic, Canada) and cDNA synthesis kit (Yekta Tajhiz Azma, Iran), respectively [13].Each qRT-PCR reaction had a final volume of 15 µL and all reactions followed a standardized thermal cycling protocol: pre-incubation at 95 °C for 600 s, followed by 40 cycles of three amplification steps at 95 °C for 20 s, 64 °C, 62 °C, and 59 °C (for rpsL, mexY, and phoP genes, respectively) for 20 s, and 72 °C for 30 s.The rpsL housekeeping gene along with P. aeruginosa ATCC 27,853 were served as the reference gene and the reference strain, respectively, and changes in gene expression levels of the target genes were calculated using the comparative Ct (2 -ΔΔCt ) method.Each experiment was replicated twice, and the interpretation of the results was conducted based on previous studies [13,14].
Based on PCR analysis, 86.4% (83/96) of the tested isolates were positive for genes encoding aminoglycosidemodifying enzymes.The most prevalent gene detected was aac(6')-Ib (82/96, 85.4%), followed by ant(2'')-Ia (18/96, 18.7%), and aph(3')-VI (3/96, 3.1%).Notably, the aac(6')-IIa gene was not identified in our study.Interestingly, among the 96 aminoglycoside-resistant P. aeruginosa isolates, 14 (14.5%) did not harbor any known aminoglycoside resistance genes.The genotypic and phenotypic profiles of aminoglycoside resistance among the 96 P. aeruginosa isolates are presented in Tables 3 and 4. Additionally, the genes responsible for methylation of the 16S rRNA in the 30 S ribosomal subunit were not found to be associated with the emergence of aminoglycoside resistance in P. aeruginosa isolates.Details of genotypic detection of aminoglycoside resistance-associated genes in P. aeruginosa clinical strains are depicted in Supplementary Figure S1.
The MexXY-OprM efflux pump and PhoPQ TCS genes were identified in all aminoglycoside-resistant P. aeruginosa isolates using the PCR analysis.However, as outlined in Table 5, the expression levels of the mexY and phoP genes, assessed via qRT-PCR, among 16 selected aminoglycoside-resistant P. aeruginosa isolates were 75% (12/16) and 87.5% (14/16), respectively.Details of amplification curves and melting peaks for each target in qRT-PCR are presented in Supplementary Figure S2.

Discussion
Since the introduction of broad-spectrum aminoglycosides in the 1940s, alongside beta-lactams and fluoroquinolones, they have remained crucial as antipseudomonal agents [13,23].However, a comprehensive analysis of our current and previous studies reveals a significant prevalence of aminoglycoside resistance among P. aeruginosa strains isolated from hospitals in Ardabil (48%), which is comparable to the resistance rates observed for betalactams such as penicillins (46.4% to 94%), carbapenems (33.3% to 66.7%), monobactams (42.9%), cephems (46.5% to 50%), and fluoroquinolones (52.4% to 76.2%) [4].This is a matter of concern and underscores the potential threat posed to the health of individuals receiving medical care at Ardabil hospitals.An important contributing factor to the high levels of aminoglycoside resistance in Ardabil hospitals is their extensive use in the treatment    of life-threatening infections caused by various organisms, including urinary tract infections, sepsis, and pneumonia [23].Therefore, it is imperative to develop strategies to optimize the use of antimicrobial agents, such as combination therapy, rather than relying solely on individual agents.Among the aminoglycosides, amikacin, gentamicin, and tobramycin are the most commonly used in clinical practice [23].Amikacin serves as an indicator antibiotic for the treatment of P. aeruginosa infections, and amikacin-resistant strains often display cross-resistance to other aminoglycosides [17].In this study, among the 86 tested amikacin-resistant P. aeruginosa strains, resistance rates to tobramycin and netilmicin were 95.3%, and 100%, respectively.It is worth noting that the average prevalence of amikacin resistance among P. aeruginosa strains in Iran is 50.6% [24].The resistance to amikacin observed in this study was higher than the rates reported in Urmia (30.7%),Hamadan (30.2%), and Zanjan (21.7%), while being lower than the rates reported in Isfahan (95.5%),Tehran (80%), Ahvaz (55.2%), and Guilan (48.8%) [24].The prevalence of amikacin-resistant P. aeruginosa strains in other countries was as follows: China (85%), the USA (6%), and 11 European countries (12.9%) [25][26][27].Differences in these results may be attributed to geographic variations, overuse of aminoglycosides in hospitals, self-medication practices without prescription, and differences in the overall health status of the populations studied.Amikacin and gentamicin, when combined with other antibiotic classes, are recommended for the treatment of infections caused by MDR Gram-negative organisms [23].Gentamicin disk diffusion and MIC breakpoints for P. aeruginosa was deleted in new CLSI breakpoint revision [11].Based on the previous version, the resistance rate to gentamicin observed in this study was similar to the national average (46% vs. 46.9%)[24].Interestingly, the prevalence of MDR strains among the 96 aminoglycoside-resistant P. aeruginosa strains was higher than the national average (94.7%)[24].In addition, it was higher than Ahvaz (91.9%),Hamadan (88.7%),Tehran (81.3%),Tabriz (68%), Zanjan (65%), Isfahan (63.1%),Urmia (56.9%),Guilan (45.5%), and Zahedan (16.4%) [24].The identification of genes encoding resistance to aminoglycosides is crucial for managing drug-resistant infections and preventing treatment failure [17].The predominant mechanism of aminoglycoside resistance involves aminoglycoside modifying enzymes [28], which was corroborated in our study, with 86.4% of the strains exhibiting this type of resistance.Among these enzymes, the aac(6')-Ib gene emerged as the most prevalent among aminoglycoside-resistant P. aeruginosa strains [28].Our findings revealed the presence of the aac(  [31].However, in our analysis, no significant correlation was observed between tobramycin resistance and the presence of aph(3')-VI (Table 4).Considering the high prevalence of aminoglycoside resistance among P. aeruginosa strains in Ardabil hospitals, largely attributed to aminoglycoside-modifying enzymes, the utilization of semisynthetic aminoglycosides to overcome common resistance mechanisms is recommended [23].One notable example of a semisynthetic aminoglycoside is plazomicin, which was approved by the FDA (Food and Drug Administration) in June 2018 for the treatment of urinary tract infections caused by certain susceptible bacteria [23].Plazomicin was specifically engineered to circumvent aminoglycoside-modifying enzymes [23].Fourteen aminoglycoside-resistant P. aeruginosa strains exhibited positive phenotypic tests but did not show the presence of genes encoding aminoglycoside-modifying enzymes.
There are two possible explanations for this observation: 1) the genes encoding aminoglycoside-modifying enzymes are typically carried on plasmids [28], and 2) other resistance mechanisms, such as 16S rRNA methylases, biofilm formation, MexXY-OprM efflux pump, and TCS, may be involved.In line with a report by Kim et al., none of the P. aeruginosa strains in our study harbored the 16S rRNA methylases gene.We speculate that this is because the 16S rRNA methylases gene is encoded on the same plasmid as the aminoglycoside-modifying enzymes [28].Decreased drug accumulation via overexpression of the MexXY-OprM efflux pump gene confers low-level intrinsic resistance to aminoglycosides in P. aeruginosa [28].As shown in Table 3, aminoglycoside-resistant P. aeruginosa strains with resistance mechanisms independent of aminoglycoside-modifying enzymes and 16S rRNA methylase exhibited biofilm production as well as overproduction of TCS and efflux pump.Studies have indicated that extracellular DNA (eDNA), a component of the biofilm matrix, is involved in aminoglycoside resistance by inducing the expression of genes regulated by the PhoPQ TCS [9].In the current study, 87.5% of aminoglycoside-resistant P. aeruginosa strains exhibited expression of the PhoPQ TCS.Some additional experiments were beyond the scope of this study and could be acknowledged as limitations but also provide opportunities for future research.These include evaluation of: 1) gene expression levels of the MexXY-OprM efflux pump and PhoPQ TCS in all aminoglycoside-resistant P. aeruginosa strains; 2) mutations in the PhoPQ TCS genes; and 3) the genetic relationship between bacterial strains isolated from different hospitals using a molecular typing method.

Conclusion
Considering the high prevalence of aminoglycoside-resistant P. aeruginosa strains with diverse resistance mechanisms in Ardabil hospitals, the following strategies are suggested to combat bacterial resistance to aminoglycosides: 1) enhancing public awareness regarding antibiotic resistance and advocating for judicious antibiotic use, 2) tailoring antibiotic prescriptions based on local antimicrobial resistance patterns and considering combination therapy when appropriate, 3) mitigating the occurrence of hospital-acquired infections through stringent adherence to infection control protocols, 4) implementing ongoing surveillance and research initiatives to monitor the prevalence and mechanisms of aminoglycoside resistance, given its plasmid-mediated nature, and 5) transitioning towards the utilization of novel antipseudomonal antibiotics, including emerging aminoglycosides, within clinical settings.

Table 3
Genotypic profiles of resistance to aminoglycosides among 96 P. aeruginosa isolates